The present invention relates to novel polypeptides having a nitrilase activity and to the genetic engineering tools for producing them, namely a DNA sequence, the expression cassettes carrying this recombinant DNA sequence, and the recombinant microorganisms (host microorganisms) containing said DNA sequence.
The present invention further relates to an enzymatic method of converting nitriles to carboxylates by means of the polypeptides according to the invention or a host microorganism containing the DNA sequence according to the invention.
A first particular application of the method of the invention is the enzymatic synthesis of ammonium adipate or ammonium 5-cyanovalerate by the hydrolysis of adiponitrile with the aid of a polypeptide or host microorganism according to the invention.
Ammonium adipate is known to be a particularly valuable product because it can be converted to adipic acid, a product which is itself widely used for the preparation of nylon 6,6.
The enzymatic hydrolysis of dinitriles has been described by numerous authors. However, the routes by which these dinitriles are hydrolyzed to organic acids are not often referred to. The theoretical hydrolysis scheme is shown in FIG. 12.
In actual fact, it is very often observed that certain routes are preferred and that certain products are not formed or else are not hydrolyzed.
Among the microorganisms for which it has been possible to demonstrate the existence of an enzymatic activity permitting this hydrolysis, there may be mentioned in particular the strains belonging to the genus Fusarium, which degrade succinonitrile and adiponitrile, although the reaction products are not indicated [Goldlust et al., Biotechnol. and Appl. Biochem., 1989, 11, 581]; the strains belonging to the genus Pseudomonas, which degrade adiponitrile [Yanase et al., Agric. Biol. Chem., 1982, 46, 2925]; and the strains belonging to the genus Rhodococcus, in particular Rhodococcus rhodochrous NCIB 11 216, which hydrolyzes adiponitrile to adipic acid [Bengis-Garber et al., Appl. Microbiol. Biotechnol., 1989, 32, II], and also Rhodococcus rhodochrous K22, whose nitrilase permits the hydrolysis of adiponitrile and glutaronitrile [Yamada et al., J. Bacteriol., 1990, 172 (9), 4807-4815], albeit with a low activity ratio compared with that for the hydrolysis of aromatic nitriles.
Consequently, it can be seen that the enzymatic hydrolysis of dinitriles is rather complex: in all cases, the first CN group is hydrolyzed by the enzyme, while the second group is not hydrolyzed at all in some cases, or else is hydrolyzed at a very low rate in other cases.